Apparatus and method for separation of liquid phases of different density and for fluorous phase organic syntheses

ABSTRACT

A simple, efficient apparatus and method for separating layers of immiscible or partially miscible liquids useful in methods of high-throughput combinatorial organic synthesis or parallel extraction of large libraries or megaarrays of organic compounds is disclosed. The apparatus and method are useful, whether as part of an automated, robotic or manual system for combinatorial organic synthesis or purification (extraction). In a preferred embodiment, an apparatus and method for separating layers of immiscible or partially miscible liquids compatible with microtiter plate type array(s) of reaction vessels is disclosed. Another application of centrifugation based liquid removal was found for washing the plates in biological assays or synthesis on modified substrates.

[0001] This application is a continuing application of U.S. S. No.60/118,377 filed Jan. 28, 1999.

FIELD OF INVENTION

[0002] The present invention relates to the field of devices and methodsfor chemical synthesis, analysis, and biological screening. Moreparticularly, the present invention relates to a simple efficientapparatus and method for separation of immiscible or partially miscibleliquid phases of different density in high-throughput, organicsynthesis, or for removal of liquid from the vessels (washing). Thepresent invention is particularly applicable for high-throughputcombinatorial synthesis of organic molecules, whether as part of anautomated or a manual procedure.

BACKGROUND OF THE INVENTION

[0003] Solid phase synthesis of organic molecules is the method ofchoice for preparation of libraries and compound megaarrays, which arecurrently being applied for screening in the quest to find new drugs orpharmaceutical lead compounds, i.e., compounds which exhibit aparticular biological activity of pharmaceutical interest, and which canserve as a starting point for the selection and synthesis of a drugcompound, which in addition to the particular biological activity ofinterest has pharmacologic and toxicologic properties suitable foradministration to animals, including humans.

[0004] Fluorous synthesis is in its principle similar to solid phasesynthesis. In fluorous synthesis the certain component of the reaction(starting material, reagent, or product) is preferentially retained inthe fluorine atoms containing phase due to its high content of fluorineatoms. Fluorous phase is usually the high density one and therefore itcan be separated as the lower phase in the multiphase system. Manualsynthesis requires repetitions of several relatively simple operationsof addition of reagents, incubation and separation of liquid phases.This character of the synthetic process renders it optimal forautomation.

[0005] Several designs of automated instruments for combinatorialsynthesis utilizing solid phase synthesis have appeared in the patentand non-patent literature. However, there is no instrument designed forthe fluorous synthesis, since the simple principle of separation ofphases by filtration is not applicable.

[0006] The productivity of automated instruments can be dramaticallyimproved by use of disposable reaction vessels (such as multititerplates or test tube arrays) into which reagents are added by pipetting,or by direct delivery from storage containers. The optimal storagevehicle is a syringe-like apparatus of a material inert to the chemicalreactants, etc., e.g., a glass syringe, allowing the storage of thesolution without any exposure to the atmosphere, and capable of servingas a delivery mechanism at the same time. See U.S. patent applicationSer. No. 08/815,975. An alternative technique based on the removal ofupper layer of liquid by suction from the surface above the separatedlayers is limited to the arrays of up to a hundred of suction needles.(For similar situation in solid phase synthesis see U.S. patentapplication Ser. No. 08/815,975). The present application is animprovement upon U.S. Pat. Nos 5,202,418, 5,338,831, 5,342,585, and U.S.patent application Ser. No. 08/815,975 which describe placement of resinin polypropylene mesh packets and removal of liquid through the openingsof these packets, or removal of the liquid from the pieces of poroustextile-like material by centrifugation, or removal of liquid phase fromthe solid phase by centrifugation of tilted plates. Liquid removal bycentrifugation was described and is the subject of several publications(see the book “Aspects of the Merrified Peptide Synthesis” by ChristianBirr in the series Reactivity and Structure Concepts in OrganicChemistry vol. 8, K. Hafner, J.-M. Lehn, C. W. Rees, P. von Rague,Schleyer, B. M. Trost, R. Zahradnik, Eds., Sringer-Verlag, Berlin,Heidelberg, N.Y., 1978, and German Patent Application P 20 17351.7, G.70 13256.8, 1970. These references describe the use of centrifugationfor liquid removal from slurry of solid phase particles in aconcentrical vessel equipped with a filtration material in its perimeterand spun around its axis. See also WO99/25470, hereby expresslyincorporated by reference in its entirety.

[0007] None of the prior art contemplates the separation of two (ormore) immiscible, or partially miscible liquids of different density byremoval of lighter layers of liquids by creation of “pockets” from whichmaterial cannot be removed by centrifugal force. This technique can beused in situations where multiplicity of products are to be extracted inparallel (e.g. in parallel purification of products of combinatorialsynthesis). However, there is a need for a simple, efficient means ofseparating liquid phases during fluorous phase synthesis of organicmolecules, particularly a method amenable to use with automated methodsfor such syntheses.

[0008] Furthermore, complete removal of the liquid from the multiplicityof vessels by spinning the array of wells attached with “reverse tilt”(tilting away from the axis of rotation) can find its application inbiological assays where fast repeated washings of surface bound reagentsor cells are required, and in applications where synthesis is donedirectly on the surface of the reaction vessels.

SUMMARY OF THE INVENTION

[0009] In accordance with the above objects, the present inventionprovides methods for elimination of a liquid phase from reaction vesselscomprising positioning a plurality of reaction vessels containing aliquid or mixture of liquids in a holder on the perimeter of acentrifuge rotor. The holder, and thus the reaction vessels, are held ina tilted position with a tilt away from the axis of rotation. The rotoris then spun at a speed that expels the liquid from the vessels. Thismethod of elimination can be used during solid-phase organic synthesis,for example synthesis of peptides or nucleic acids. Optionally, thesesteps can be repeated. Similarly, the reaction vessels may be containedin a microtiter plate or the reaction vessel may be a microtiter plate.

[0010] The expelled liquid can be collected in a collection pocket inthe holder having a volume sufficient to collect and retain any liquidexpelled from the vessels, or can be collected in a waste reservoir inor outside of the centrifuge.

[0011] In an additional aspect, the invention provides methods ofsynthesis of compounds comprising providing a reaction vessel containinga first building block coupled to the vessel itself. The vessel is thenpositioned in a holder on the perimeter of a centrifuge rotor. A secondbuilding block is added to the vessel under conditions that allow thecoupling of the first and second building blocks, and the rotor is spunat a speed sufficient to expel the liquid from the vessel. Optionaladditional steps of repeating the procedure or washing steps can also beincluded. The building blocks can include amino acids and nucleosides.

[0012] In a further aspect, the invention provides methods forseparating at least two immiscible or partially miscible liquidscomprising positioning a plurality of reaction vessels containing theliquids in a holder on the perimeter of a centrifuge rotor. The rotor isthen spun at a speed such that the lower layer of the multiphase systemis retained in a “pocket” of the vessels and the upper layer is expelledfrom the vessels.

[0013] In an additional aspect, the invention provides apparatuscomprising a centrifuge comprising a rotor designed to hold reactionvessels at a tilt away from the axis of rotation and a waste reservoirconnected to the centrifuge to hold liquids expelled from the reactionvessels. In one embodiment, the waste reservoir is connected to thecentrifuge with a tube. The apparatus may optionally comprise a liquiddistribution system and a computer processor.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The present invention can be understood more completely byreference to the following detailed description, examples, appendedclaims and accompanying figures.

[0015]FIGS. 1A, 1B and 1C illustrate separation of two immiscible orpartially miscible liquid phases in a “pocket” of the vessels andexpulsion of upper liquid layer achieved according to the method of theinvention. FIG. 1A illustrates the lower liquid phase and upper liquidphase in the vessel prior to centrifugation. FIGS. 1B and 1C illustratethe pocket containing retained lower liquid phase layer during spinning(and removal of the upper liquid layer).

[0016]FIGS. 2A, 2B and 2C illustrate the path of liquid removed from avessel, such as a well of a microtiter plate by centrifugation. Thestraight lip at the upper end of each well of the microtiter plateprevents the liquid from entering the well closer to the edge of acentrifugal plate—this well is higher and the lip wall is tilted in thedirection to the bottom of the plate. The large arrow represents thevector resulting from centrifugal and gravitational forces. The smallarrow with thin trailing line illustrates the direction of the flow ofliquid removed.

[0017]FIGS. 3A and 3B illustrate an alternative embodiment of theinvention in which a vessel having a lip facing inward when spunaccording to the method of the invention “creates” a “pocket” in whichthe lower liquid phase is retained.

[0018]FIGS. 4A, 4B and 4C illustrate the situation in which wells aretilted in “reverse” tilt and no “pocket” is formed duringcentrifugation. The result is the complete removal of all liquid fromthe wells.

[0019]FIG. 5 shows the UV spectra of wells before and after two steps ofparallel extraction proving complete elimination of contamination byaromatic hydrocarbon.

5. DETAILED DESCRIPTION OF THE INVENTION

[0020] For clarity of disclosure, and not by way of limitation, thedetailed description of this invention is presented herein with respectto figures that illustrate preferred embodiments of elements of thisinvention. However, this invention includes those alternativeembodiments of these elements performing similar functions in similarmanners that will be apparent to one skilled in the art from theentirety of the disclosure provided. In addition, all referencesdisclosed herein are incorporated by reference in their entirety.

[0021] The present invention is based on a discovery of a simpleefficient means for separation of two (or more) immiscible, or partiallymiscible liquids of different density, e.g., removal of upper layer orlayers of liquid from lower layer, used for parallel extraction and/orin fluorous phase organic syntheses. In one embodiment of the invention,the fluorous phase organic synthetic protocol utilizes widely available,disposable reaction vessel arrays, such as microtiter style plates. Inan alternative embodiment of the invention, the synthetic protocolutilizes a vessel with a lip facing inward (see FIG. 2) spun around itsaxis to create a “pocket” in which the lower layer of the multiphasesystem is retained. According to the present invention, however, anyvessel or array of vessels or plurality of arrays of vessels which canbe placed in a tilted position on the perimeter of a centrifuge, can beused in the method of the invention. The method of the invention forseparating of two (or more) immiscible, or partially miscible liquids ofdifferent density during a parallel extraction and/or in fluorous phaseorganic synthetic process comprises: (1) positioning a reaction vesselor an array of reaction vessels, such as a microtiter plate having anarray of reaction wells, said vessel(s) containing a multilayer systemof liquid phases, on the perimeter of a centrifuge rotor in a tiltedposition; and (2) spinning the rotor of the centrifuge at a speed sothat the lower layer fills a “pocket” of the vessels and the upperliquid phases are expelled from the vessels. In one embodiment of theinvention, the rotor is spun at a speed so that the centrifugal force onthe radius corresponding to the reaction vessels which are closest tothe axis of rotation is significantly greater than the force of gravity,and the lower layer forms a “pocket” of the vessels and the upper layersare expelled from the vessels. The volume of a “pocket” is determinedby: (i) the degree of the tilt, (ii) the speed of rotation, and (iii)the distance of the particular reaction vessel from the axis ofrotation. The appropriate combination of these factors determines thevolume of residual liquid retained in the pocket and thereforecompleteness of upper layer removal. However, since it is desired thatall reaction vessels in a multivessel arrangement of a reaction block(such as a microtiter plate) should undergo the removal of the upperlayers of liquid to the same degree, it is important that the angle ofthe liquid surface in the “pocket” of the reaction vessels during thecentrifugation is as close to 90 degrees with respect to the center ofrotation as possible. In one embodiment, the removed liquid phase iscollected on the wall of the centrifuge. In an alternative embodiment,the removed liquid phase is collected in a “collecting pocket” or aseries of “collecting pockets” attached at the perimeter of thecentrifuge rotor. The apparatus of the invention comprises a holderadapted to attaching a reaction vessel or an array of reaction vessels,e.g., a microtiter plate, to a rotor of a centrifuge, said holdercomprising one or more indentations or groves designated “collectingpockets” positioned along one side of said holder said collectingpockets having a volume sufficient to collect and retain any liquidexpelled from the reaction vessels, e.g./ the wells of the microtiterplate, when the holder and attached reaction vessels are spun by thecentrifuge rotor. According to the invention, the holder can hold asingle or individual microtiter plate or a plurality of microtiterplates, each plate comprising an array of vessels. One or more of theholders can be attached to the rotor of a centrifuge. In anotherembodiment, the apparatus of the invention is an automated integratedapparatus or system for parallel extraction and/or for fluorous phasechemical synthesis, comprising: (a) a centrifuge in which an array ofreaction vessels suitable for parallel extraction and/or for fluorousphase organic synthesis can be spun in a tilted position; (b) a liquiddistribution device; and (c) a computer for processing a program ofinstructions for addition of liquid phase to and removal, viacentrifugation, of upper layer liquid phase from the reaction vesselsaccording to the program.

[0022] In general, the methods and apparatus of the invention find usein combinatorial chemical synthesis. By way of introduction,combinatorial chemistry synthesis protocols prescribe the stepwise,sequential addition of building blocks to intermediate and/or partiallysynthesized intermediate compounds in order to synthesize a finalcompound. In solid-phase synthesis, final compounds are synthesizedattached to solid-phase supports that permit the use of simplemechanical means to separate intermediate, partially-synthesizedintermediate compounds between synthetic steps. Typical solid-phasesupports include beads, including microbeads, of 30 microns to 300microns in diameter, which are functionalized in order to covalentlyattach intermediate compounds (or final compounds), and made of, e.g.,various glasses, plastics, or resins.

[0023] WO 99/25470, hereby incorporated by reference, describes the useof a centrifuge in solid-phase synthetic reactions, wherein particles ofsolid phase (microbeads) are contained in reaction vessels such asmicrotiter plates. Synthesis is achieved by the stepwise addition of“building blocks” of the biopolymer, followed by centrifugation thatdrives the liquid phase out of the reaction vessels yet traps the solidphase in “pockets”.

[0024] This general idea can be applied to differential phase syntheticreactions as well. While described for fluorous synthesis, one of skillin the art will appreciate that these techniques work for otherphase-dependent synthetic methods as well.

[0025] The principle of fluorous phase synthesis is very similar tosolid phase synthesis. In fluorous phase synthesis one of the reagentsis attached to a high fluorine content block (“fluorous tail”), whichassures that this reactant will always have a tendency to stay influorocarbon based solvent layer. Due to the fact that some fluorocarbonbased solvents are not miscible (or only partially miscible) with bothorganic solvents and water and that this phase is in most cases thephase with the highest density, its properties can be used to mimic thesolid phase principle of synthesis. Due to the fact that fluorous phasesynthesis technology is at its very early stage of development, thegeneral process for application in the combinatorial synthesis can beonly speculated on. Fluorous phase combinatorial synthesis shouldproceed according to the following steps. In a first step, reactionvessels are charged with a fluorous phase, e.g. benzotrifluoride, andthe first component of the synthesis (sometimes referred to herein as“the first building block”) with attached “fluorous tail” (blockcontaining high proportion of fluorine atoms) is delivered to all wells.Subsequently, a plurality of building block addition steps areperformed, all of which involve repetitive execution of the followingsubsteps, and in a sequence chosen to synthesize the desired compound.First, a sufficient quantity of a solution containing the building blockmoiety (e.g. the “second building block”, “third building block”, etc.)selected for addition is accurately added to the reaction vessels sothat the building block moiety is present in a molar excess to theintermediate compound (compound with fluorous tail). The reaction istriggered and promoted by activating reagents and other reagents andsolvents, which are also added to the reaction vessel. The reactionvessel is then incubated at a controlled temperature for a time,typically between 5 minutes and 24 hours, sufficient for the buildingblock addition reaction or transformation to go to substantialcompletion. Optionally, during this incubation, the reaction vessel canbe intermittently agitated or stirred. Finally, in a last substep ofbuilding block addition, the reaction vessel containing the fluorousphase with intermediate compound attached to fluorous tail is preparedfor addition of the next building block by removing the reaction fluidand thorough washing and reconditioning the fluorous phase by washing(repetitive addition and removal by centrifugation) with water and/ororganic solvents. The limitation is that the fluorous phase must formalways the lower phase of the system, which can be multilayer(multiphase).

[0026] Washing typically involves three to seven cycles of adding andremoving a wash solvent. Optionally, during the addition steps, multiplebuilding blocks can be added to one reaction vessel in order tosynthesize a mixture of compound intermediates attached to one fluoroustail, or alternatively, the contents of separate reaction vessels can becombined and partitioned in order that multiple compounds can besynthesized in one reaction vessel.

[0027] After the desired number of building block addition steps, thefinal compound is present in the reaction vessel attached to thefluorous tail. The final compounds can be utilized either directlyattached to the fluorous tail, or alternatively, can be cleaved from thefluorous tail and purified by extraction. Examples of fluorous phasesynthetic protocols can be found in the following references: Curran, D.P. (1996) Combinatorial organic synthesis and phase separation: Back tothe future. Chemtracts: Org. Chem., 9, 75-87; Curran, D. P., & Hoshino,M. (1996) Stille couplings with fluorous tin reactants: Attractivefeatures for preparative organic synthesis and liquid-phasecombinatorial synthesis. J. Org. Chem., 61, 6480-6481; Curran, D. P.(1998) Fluorous synthesis: An alternative to organic synthesis and solidphase synthesis for the preparation of small organic molecules. Canc. J.Sci. Amer., 4, S73-S76; Curran, D. P. (1998) Strategy-level separationsin organic synthesis: From planning to practice. Angew. Chem. Int. Ed.,37, 1175-1196; Ogawa, A., & Curran, D. P. (1997) Benzotrifluoride: Auseful alternative solvent for organic reactions currently conducted indichloromethane and related solvents. J. Org. Chem., 62, 450-451;Studer, A., Jeger, P., Wipf, P., & Curran, D. P. (1997) Fluoroussynthesis: Fluorous protocols for the Ugi and Biginelli multicomponentcondensations. J. Org. Chem., 62, 2917-2924; Studer, A., & Curran, D. P.(1997) A strategic alternative to solid phase synthesis: Preparation ofa small isoxazoline library by “fluorous synthesis”. Tetrahedron, 53,6681-6696; Studer, A., Hadida, S., Ferritto, R., Kim, S. Y., Jeger, P.,Wipf, P., & Curran, D. P. (1997) Fluorous synthesis: A fluorous-phasestrategy for improving separation efficiency in organic synthesis.Science, 275, 823-826. As for all the references herein, these areexpressly incorporated by reference in their entirety.

[0028] Accordingly, in a preferred embodiment, the invention providesmethods for separation of immiscible or partially miscible liquid phasesof different density during a parallel extraction, including a fluorousphase organic synthetic process. The methods comprise: (1) positioning areaction vessel or one or more arrays of reaction vessels, such as oneor more microtiter plates, said vessels containing an immiscible orpartially miscible liquid phases of different density on the perimeterof a centrifuge rotor in a tilted position; and (2) spinning the rotorof the centrifuge at a speed so that the lower phase is retained in a“pocket” of the vessels and the upper liquid phase(s) is (are) expelledfrom the vessels. In the case where only one row of vessels is placed atthe perimeter of the centrifuge rotor, the ratio of centrifugal forceversus gravitation determines the volume of the “pocket” used for theseparation of liquid phases in all vessels and even very low ratio (suchas 1:1) can be successfully used. The important factor is only thereproducibility of the speed of centrifugation.

[0029] In one embodiment of the invention, the rotor of the centrifugeis spun at a speed so that the centrifugal force on the radiuscorresponding to the reaction vessels which are closest to the axis ofrotation is significantly greater than the force of gravity so that thelower liquid phase is retained in a “pocket” of the vessels and theupper liquid phase(s) is (are) expelled from the vessels. The volume ofa “pocket” is determined by: (i) the degree of the tilt, (ii) the speedof rotation, and (iii) the distance of the particular reaction vesselfrom the axis of rotation. Since it is desired that all reaction vesselsin a multivessel arrangement or array of vessels (such as a microtiterplate) should undergo the removal of the upper liquid phase to the samedegree, it is important that the angle of the liquid surface in the“pocket” of the reaction vessels during the centrifugation is as closeto 90 degrees with respect to the axis of rotation as possible. As usedin the present application, the term “significantly greater than theforce of gravity” is intended to mean that the force is at least about 5to 300×G, preferably about 10 to 300×G, and even more preferably about100 to 300×G. In other words, the centrifuge is spun at a speed so thatthe ratio of the centrifugal force to gravity, i.e., the RelativeCentrifugal Force (RCF) is at least about 5 to 300, preferably about 10to 300, and more preferably about 100 to 300. Values of RCFsignificantly greater than 1 are required if individual vessels areplaced at different distances from the center of rotation. To achieveuniform distribution of liquid in all vessels it is important to removeas much as possible of the upper liquid phase from all wells. Thetheoretical value of an angle of liquid surface achievable in thecentrifuge versus liquid in nondisturbed state is 90 degrees. Thisrequires a value of the above mentioned ratio (RCF) reaching infinity.For practical reasons, the difference between 89 degrees (ratio 100:1)or 85 degrees (ratio 18:1) may be acceptable. Acceptability of thisvalue depends on the degree of the tilt determining the absolute valueof the “pocket” volume. The greater the tilt, the bigger the “pocket”volume, and the bigger the tolerance to the different ratio values atdifferent radiuses. The maximal possible value of the tilt in “fixedtilt” centrifuges is 45 degrees, however, this tilt is completelyimpractical because the maximal volume of liquid in the well is equal tothe volume of the theoretical “pocket”.

[0030] Higher tilt is possible in the case of “dynamically adjustabletilt” centrifuges (centrifuges in which plate is horizontal instandstill state and “swings out” to a limited position duringrotation). According to one mode of one embodiment of the method of theinvention, when the reaction vessels used are one or more arrays ofregular wells in a microtiter plate, the rotor of the centrifuge is spunat a speed so that the centrifugal force on the radius of wells closestto the axis of rotation is about 5 to 300×G, preferably about 10 to300×G, and more preferably about 100 to 300×G; and the angle of tilt ofthe plate is about 1 to 45, preferably 5 to 20, and more preferably 5 to15 degrees. According to another mode of this embodiment of the methodof the invention, when the reaction vessels used are one or more arraysof microwells in a microtiter plate, the rotor of the centrifuge is spunat a speed so that the centrifugal force on the radius of wells closestto the axis of rotation is about 5 to 300×G, preferably about 10 to300×G, and more preferably about 100 to 300×G and the angle of tilt ofthe plate is about 2 to 25, preferably 2 to 10 degrees. In oneembodiment, the upper liquid phase is collected on the wall of thecentrifuge. In an alternative embodiment, the upper liquid phase iscollected in a “collecting pocket” or a series of “collecting pockets”.FIG. 1 illustrate retention of lower liquid layer in a “pocket” of thevessels and expulsion of upper liquid layer achieved according to themethod of the invention. FIG. 2 illustrates the path of upper liquidlayer removed from a vessel, such as a well of a microtiter plate bycentrifugation. The straight lip at the upper end of each well of themicrotiter plate prevents the liquid from entering the well closer tothe edge of a centrifugal plate—this well is higher and the lip wall istilted in the direction to the bottom of the plate. The large arrowrepresents the vector resulting from centrifugal and gravitationalforces. The small arrow with thin trailing line illustrates thedirection of the flow of liquid removed from the reaction vessels. FIG.3 illustrates an alternative embodiment of the invention in which avessel having a lip facing inward when spun according to the method ofthe invention “creates” a “pocket” in which the lower liquid phase isretained. The left portion of FIG. 1 illustrates the lower liquid phaseand upper liquid phase in the vessel prior to centrifugation. The rightportion of FIG. 1 illustrates the pocket containing retained lowerliquid phase layer during spinning (and removal of the upper liquidlayer). As detailed above, a single reaction vessel, a single microtiterplate or a plurality of microtiter plates can be used in the process ofthe present invention.

[0031] In addition to differential phase synthesis, the presentinvention finds use in “reverse tilt” synthesis reactions. In thisembodiment, the reaction vessels, for example in a microtiter plateformat, are tilted in the direction away from the axis of rotation: thatis, the open end of the reaction vessel is pointed away from the axis ofrotation. The negative (or “reverse”) tilt (tilt in the direction awayfrom the axis of rotation) allows for the removal of all liquid contentof the well. This may be done for a variety of reasons. In a firstembodiment, this may be applicable for washing of wells, e.g. inbiological assays when binding to the surface (or modified surface) isstudied and removal of the excess of the reagents by repetitive washingis required.

[0032] A preferred embodiment for the use of “reverse” tilt is thesituation wherein the synthesis is performed on material firmly attachedto the well; that is, the material will not be expelled from thereaction vessel under the centrifugation conditions used in the process.In a preferred embodiment, the material can be a “tea-bag” type ofmaterial filled with the solid support on which the synthesis is done,or a textile like material; for examples of these supports see U.S. Pat.No. 5,202,418, hereby expressly incorporated by reference.

[0033] In a preferred embodiment, the synthesis is performed on one ormore modified surface(s) of the reaction vessel itself. In thisembodiment, rather than use a solid support such as a microbead or adense phase as the support for a synthetic reaction, the actual surfaceof the reaction vessel is used as the solid phase for syntheticreactions; liquid reagents are added, reacted, and then the residualliquid is removed via centrifugation. That is, the reaction vessel, suchas a microtiter plate, may be functionalized as a solid support for thesynthesis. In this embodiment, the reaction vessel may be any materialthat can be modified to allow synthesis; possible materials forsubstrates include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses, andplastics, and a variety of other polymers.

[0034] The functionalization of solid support surfaces such as certainpolymers with chemically reactive groups such as thiols, amines,carboxyls, etc. is generally known in the art. Some examples of thesesurface chemistries for subsequent addition of building blocks include,but are not limited to, amino groups including aliphatic and aromaticamines, carboxylic acids, aldehydes, amides, chloromethyl groups,hydrazide, hydroxyl groups, sulfonates and sulfates.

[0035] These functional groups can be used to add any number ofdifferent building block moieties to the vessels, generally using knownchemistries, including, but not limited to the use ofamino-functionalized supports, sulfhydryl linkers, etc. There are anumber of sulfhydryl reactive linkers known in the art such as SPDP,maleimides, α-haloacetyls, and pyridyl disulfides (see for example the1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Similarly, amino groups on the building blocks and on the surface can beattached using linkers; for example, a large number of stablebifunctional groups are well known in the art, includinghomobifunctional and heterobifunctional linkers (see Pierce Catalog andHandbook, pages 155-200). In an additional embodiment, carboxyl groups(either from the surface or from the building block) may be derivatizedusing well known linkers (see the Pierce catalog). For example,carbodiimides activate carboxyl groups for attack by good nucleophilessuch as amines (see Torchilin et al., Critical Rev. TheraDeutic DrugCarrier Systems, 7(4):275-308 (1991), expressly incorporated herein). Inaddition, preferred methods include systems that allow post-synthesiscleavage from the reaction vessels.

[0036] As will be appreciated by those in the art, the functionalizationwill depend on the synthesis done, as outlined below.

[0037] As will be appreciated by those in the art, in the reverse tiltembodiments, virtually any solid phase synthesis reaction may be done.Preferred embodiments include organic syntheses, including, but notlimited to, peptide synthesis, nucleic acid synthesis, and smallmolecule synthesis.

[0038] In a preferred embodiment, peptides are synthesized. By “peptide”herein is meant at least two amino acids joined via a peptide bond. Thepeptide may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. The side chains may be ineither the (R) or the (S) configuration. In the preferred embodiment,the amino acids are in the (S) or L-configuration. If non-naturallyoccurring side chains are used, non-amino acid substituents may be used,for example to prevent or retard in vivo degradations.

[0039] The stepwise solid phase synthesis of peptides is well known. Anexemplary solid-phase combinatorial protocol is that for the synthesisof peptides attached to polymer resin, which proceeds according to Lamet al., 1991, Nature 354:82-84; U.S. Pat. No. 5,510,240; Lam et al.,1994, Selectide technology: Bead-binding screening. Methods: A Companionto Methods in Enzymology 6:372-380. Another exemplary protocol is thatfor the synthesis of benzodiazepine moieties, which proceeds accordingto Bunin et al., 1992, J. Amer. Chem. Soc., 114:10997-10998 and U.S.Pat. No. 5,288,514. Also, for protocols for the addition ofN-substituted glycines to form peptoids, see, e.g., Simon, et al., 1992,Proc. Natl. Acad. Sci. USA, 89:9367-9371; Zuckermann et al., 1992, J.Amer. Chem. Soc., 114:10646-10647; WO PCT94/06,451 to Moos et al.;Approaches for synthesis of small molecular libraries were recentlyreviewed by, e.g., Krchnak and Lebl, 1996, Molecular Diversity,1:193-216; Ellman, 1996, Account. Chem. Res., 29:132-143; Armstrong etal., 1996, Account. Chem. Res., 29:123-131; Fruchtel et al., 1996,Anaew. Chem. Int. Ed., 35:1742; Thompson et al., 1996, Chem. Rev.,96:555-600; Rinnova et al., 1996, Collect. Czech. Chem. Commun., 61:171-231; Hermkens et al., 1996, Tetrahedron, 52:45274554. Exemplarybuilding blocks and reagents are amino acids, nucleosides, other organicacids, aldehydes, alcohols, and so forth, as well as bifunctionalcompounds, such as those given in Krchnak and Lebl, 1996, MolecularDiversity, 1:193-216.

[0040] In a preferred embodiment, the methods and compositions of theinvention are used to synthesize nucleic acids. By “nucleic acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, as outlined below, nucleic acid analogs are included that mayhave alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to increasethe stability and half-life of such molecules in physiologicalenvironments.

[0041] As will be appreciated by those in the art, all of these nucleicacid analogs may find use in the present invention. In addition,mixtures of naturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

[0042] The nucleic acids may contain any combination of deoxyribo- andribo-nucleotides, and any combination of bases, both naturally occurringand synthetic, including uracil, adenine, thymine, cytosine, guanine,inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. Apreferred embodiment utilizes isocytosine and isoguanine in nucleicacids designed to be complementary to other probes, rather than targetsequences, as this reduces non-specific hybridization, as is generallydescribed in U.S. Pat. No. 5,681,702. As used herein, the term“nucleoside” includes nucleotides as well as nucleoside and nucleotideanalogs, and modified nucleosides such as amino modified nucleosides orphosphoramidite nucleosides. In addition, “nucleoside” includesnon-naturally occurring analog structures. Thus for example theindividual units of a peptide nucleic acid, each containing a base, arereferred to herein as a nucleoside.

[0043] The stepwise synthesis of nucleic acids is well known, andgenerally involves the stepwise addition of protected, activatednucleoside monomers to a solid support, followed by deprotection stepsand washing steps. See generally Gait, Oligonucleotide Synthesis: APractical Approach, IRL Press, Oxford, UK 1984; Eckstein, incorporatedby reference. This is generally done either with phosphoramidite orH-phosphonate nucleosides. This is generally done in one of two ways.First, the 5′ position of the ribose is protected with4′,4-dimethoxytrityl (DMT) followed by reaction with either2-cyanoethoxy-bis-diisopropylaminophosphine in the presence ofdiisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

[0044] In a preferred embodiment, the reverse tilt method is used tosynthesize small organic molecules. As will be appreciated by those inthe art, the literature contains numerous examples of the synthesis of avariety of small molecules, particularly libraries of small molecules onsolid-phase supports; see for example Pavia et al. Bioorganic &Medicinal Chemistry 1996 4(5):659-666; Liskamp et al., Bioorganic &Medicinal Chemistry 1996 4(5):667-672; Tong et al., Bioorganic &Medicinal Chemistry 1996 4(5):693-698; Houghten et al., Bioorganic &Medicinal Chemistry 1996 4(5):709-715, Freier et al., Bioorganic &Medicinal Chemistry 1996 4(5):717-725; Bolton et al., TetrahedronLetters 1996 37(20) 3433-3436, all of which are hereby expresslyincorporated by reference.

[0045] In addition, for all the reverse tilt embodiments herein, it maybe desirable to use linkers to attach the first building blocks to thesurface.

[0046] Accordingly, the invention provides methods of synthesis usingreverse tilt centrifugation. In this embodiment, a reaction vessel orarray of vessels are provided that comprise either a pre-functionalizedfirst building block or the chemistry to attach the first building blockof the molecule to be made. Subsequently, a plurality of building blockaddition steps are performed, all of which involve repetitive executionof the following substeps, and in a sequence chosen to synthesize thedesired compound. First a sufficient quantity of a solution containingthe building block moiety selected for addition is accurately added tothe reaction vessels so that the building block moiety is present in amolar excess to the intermediate compound. The reaction is triggered andpromoted by activating reagents and other reagents and solvents asneeded, which are also added to the reaction vessel. The reaction vesselis then incubated at a controlled temperature for a time, typicallybetween 5 minutes and 24 hours, sufficient for the building blockaddition reaction or transformation to go to substantial completion.Optionally, during this incubation, the reaction vessel can beintermittently agitated or stirred. Finally, in a last substep ofbuilding block addition, the reaction vessel is prepared for addition ofthe next building block by removing the reaction fluid using the“reverse tilt” centrifugation steps outlined herein and thorough washingand reconditioning as needed. Washing typically involved three to sevencycles of adding and removing a wash solvent. Optionally, during theaddition steps, multiple building blocks can be added to one reactionvessel in order to synthesize a mixture of compound intermediatesattached to one reaction vessel. After the desired number of buildingblock addition steps, the final compound is present in the reactionvessel. It can then be optionally cleaved from the reaction vesselsupport; alternatively, the reaction vessels themselves can be used insubsequent reactions. A variety of exemplary reactions are outlined inWO99/25470, hereby incorporated by reference, and include reactions forpeptide and synthetic peptides, benzodiazepine and derivatives,peptoids, N-substituted polyamide monomers. Exemplary building blocksand reagents are amino acids, nucleic acids, other organic acids,aldehydes, alcohols, and so forth, as well as bifunctional compounds.

[0047] The present invention also provides apparatus for organicsynthesis as outlined herein. The apparatus of the invention comprise avariety of components, including a centrifuge and a rotor. In general,the rotor comprises at least one holder, and preferably a plurality ofholders, that each will hold at least a first reaction vessel, andpreferably a plurality of reaction vessels.

[0048] As will be appreciated by those in the art, the reaction vesselscan be configured in a variety of ways. In a preferred embodiment, thereaction vessels are in the form of an array of vessels such as amicrotiter plate that contains the individual reaction vessels.Particularly preferred configurations are 96-well and 384-wellmicrotiter plates.

[0049] As will be appreciated by those in the art, one of the importantaspects of the invention is that one or more liquid phases are expelledfrom the reaction vessels upon centrifugation. Accordingly, there aretwo main ways the system may be configured to allow the collection ofthe expelled liquids.

[0050] In a preferred embodiment, the holders adapted to attaching amicrotiter plate to a centrifuge rotor can have or comprise a series ofcollecting pockets to collect and retain the liquid expelled from thevessels during centrifugation. These collecting pockets can comprise oneor more indentations or grooves having a volume sufficient to collectand retain any expelled liquid.

[0051] In an alternative preferred embodiment, the holder does not havecollecting pockets. In the latter situation, the liquid expelled isdeposited on the walls of the centrifuge. In this embodiment, thecentrifuge is configured such that there is a collecting pocket orreservoir, generally in the bottom of the centrifuge, such that gravityflow of the expelled liquids causes the liquids to pool in the pocket.This may be periodically emptied as needed, and can comprise a port orvalve that allows drainage.

[0052] Alternatively, the centrifuge is configured to have a tube orpipe leading to a waste reservoir; this tube is also generally at thebottom of the centrifuge. The gravity flow of the expelled liquids canthen lead to collection of the waste outside the centrifuge.

[0053] In a preferred embodiment, the holder(s) hold the reactionvessels in a tilted position. The holder(s) may either hold one or moreof the reaction vessels in a fixed tilted position or in a position inwhich the angle of tilt can be changed flexibly.

[0054] As outlined herein, the tilt of the rotor can be towards the axisof rotation, resulting in the retention of a phase of the reaction.Alternatively, when the synthetic reaction is done on material firmlyattached to the reaction vessel (e.g. with a force such that it will notbe expelled during the centrifugation) or when the reaction is done onthe reaction vessel itself, the tilt of the rotor can be away from theaxis of rotation (“reverse tilt”) as described herein.

[0055] In a preferred embodiment, each holder contains only one set orarray of reaction vessels. Thus, for example, the holder may containgrooves or rails to position the reaction vessels, e.g. microtiterplate, in the holders. Alternatively, the reaction vessels may be“stacked” or “layered”. However, placing single sets or arrays ofreaction vessels such as individual microtiter plates on the centrifugeperimeter has an advantage of simple interfacing with liquiddistribution automats (such as Packard Can berra, Tecan, Hamilton, andothers). A liquid distribution device can be placed onto the top of acentrifugal synthesizer. Particularly preferred are liquid distributionsystems for simultaneous dispensing in a format that fits the reactionvessel configuration; for example, when the reaction vessels are in theform of a 96 well microtiter plate, the liquid distribution system ispreferably a 96 channel device.

[0056] The liquid distribution system can also comprise a set ofreservoirs and tubes for delivery; for example, the liquid distributionsystem can have a 96 channel liquid distributor that can deliver solventor solutions of reagents from different bottles into the platepositioned under the needles of the distributor. For example, fornucleic acid synthesis, preferred embodiments include separate reagentbottles for each nucleoside.

[0057] In a preferred embodiment, the liquid distribution system is anintegrated system; that is, the liquid is distributed into the reactionvessels when they are present in the centrifuge; the reaction vesselsare not removed from the centrifuge for addition of reagents. Ingeneral, the liquid distribution system is an integral part of thecentrifuge; the liquid is delivered without removing the lid of thecentrifuge.

[0058] In addition, the apparatus of the invention can further comprisea processor or computer to control the synthesis of the moieties. Forexample, a computer may be used that processes a program of instructionsof stepwise additions of liquid phases, reagents, solvents, washes, etc.to the reaction vessels, followed by centrifugation steps for removal ofliquids from the reaction vessels. Thus, the present invention providesmethods executed by a computer under the control of a program, thecomputer including a memory for storing the program. The program isdirected to the addition of reagents to the reaction vessels using theliquid distribution system, allowing incubation as needed, and removingunreacted reagents and liquid by centrifugation for a defined time at adefined speed, with wash steps and repetition as required.

[0059] In addition to the components mentioned above, the centrifuge mayalso comprise additional components. For example, the centrifuge cancomprise a sensor to signal the computer and liquid distribution systemwhen a set of reaction vessels in a particular orientation, and a motorto rotate the rotor into the correct orientation for liquid delivery,also in control of the computer. Furthermore, in the case of adjustabletilt holders or rotors, the centrifuge can utilize a control and asensor to control the degree of tilt.

[0060] The integrated device is useful as a “centrifugation synthesizer”for fluorous phase synthetic processes.

[0061] The methods and apparatus of the invention find use in a numberof applications, as outlined herein.

[0062] In a preferred embodiment, the methods and apparatus of thepresent invention are advantageously useful for the manual or automatedpreparation of combinatorial libraries or megaarrays of compounds byfluorous phase organic synthesis. As is well known to those skilled inthe art, such combinatorial libraries or megaarrays have numerous uses,in particular, for the selection of pharmaceutical lead compounds, forthe optimization of pharmaceutical lead compounds and for theidentification and/or isolation of pharmaceutical drugs. The methods andapparatus of the invention for liquid/liquid phase separation can alsoadvantageously be used for parallel extraction and purification ofcompound arrays synthesized or obtained by other methods. Otherapplications in analytical chemistry (extraction, desalting or othermeans of parallel preparations of samples), biochemistry (parallelprocessing of samples) are envisioned.

[0063] The use of complete removal of liquid from the arrays of vesselscan be applied in the biological screening where binding to the surfaceof the vessels (modified surface by attached reagent or cell culture) isinvestigated. In this case the tilt of the vessel during thecentrifugation is “reversed”, i.e. no “pocket” is formed during thecentrifugation. In this case, for example, any number of binding assaysmay be done. For example, ELISA type assays are frequently done in amicrotiter plate format, where antibodies are attached using a varietyof known chemistries. The addition of sample(s) and additional reagentcomponents, with washing as required, may utilize the present invention.Furthermore, in this embodiment, the apparatus may comprise additionalcomponents such as fluorescence readers.

[0064] Similarly, reverse tilt reactions can be used in syntheticreactions as outlined above, with particular emphasis on nucleic acidand peptide synthesis.

EXAMPLE Removal of Upper Layer Liquid Phase Without Transfer of LowerLayer Liquid Phase

[0065] Ten percent solution of ethanol in water saturated with toluenewas distributed into wells of microtiterplate (40 uL per well). Ethylacetate (150 uL) was repeatedly distributed into the wells andmicrotiterplates were shaken for 1 minute and centrifugated in tiltedarrangement. FIG. 5 shows UV spectra of wells before and after two stepsof parallel extraction proving complete elimination of contamination byaromatic hydrocarbon.

1-32. (cancelled)
 33. A method for elimination of a liquid phase from areaction vessel, said method comprising: (a) providing a centrifugerotor having a reaction vessel and a material firmly attached to saidreaction vessel, said material functionalized in order to attachcompounds thereto; (b) filling said reaction vessel with a liquid; and(c) spinning said centrifuge rotor at a speed so that the liquid isexpelled from said vessels such that said material is not expelledduring centrifugation.
 34. The method according to claim 33, wherein themethod of elimination is employed during solid-phase organic synthesis.35. The method according to claim 34, wherein said solid-phase organicsynthesis is synthesis of peptides.
 36. The method according to claim34, wherein said solid-phase organic synthesis is synthesis of nucleicacids.
 37. The method according to claim 33, further comprisingrepeating steps (a), (b) and (c), whereby an organic molecule issynthesized.
 38. The method according to claim 33, wherein said reactionvessel comprises at least one microtiter plate.
 39. The method accordingto claim 33, wherein said rotor comprises a plurality of holders. 40.The method according to claim 33, wherein said material comprises abead.
 41. The method according to claim 40, said bead comprises amicrobead having a diameter of approximately 30 to 300 microns.
 42. Themethod according to claim 33, wherein an open end of said reactionvessel is pointed away from the axis of rotation in step (c).
 43. Amethod of synthesizing compounds, said method comprising: (a) providinga centrifuge rotor having a reaction vessel and a first building blockcoupled to said reaction vessel; (b) adding a second building block tosaid reaction vessel; and (c) spinning said centrifuge rotor at a speedsufficient to expel said liquid from said vessel.
 44. The methodaccording to claim 43, wherein an open end of said reaction vessel ispointed away from the axis of rotation in step (c).
 45. The methodaccording to claim 43, wherein said reaction vessel is part of amicrotiter plate.
 46. The method according to claim 43, furthercomprising repeating steps (b) and (c) whereby an organic moiety issynthesized.
 47. The method according to claim 43, further comprisingwashing said solid support prior to adding additional building blocks.48. The method according to claim 43, wherein said building blocks areamino acids.
 49. A method according to claim 43, wherein said buildingblocks are nucleosides.
 50. A method according to claim 43, wherein saidfirst building block is coupled to said vessel by material firmlyattached to said reaction vessel.
 51. The method according to claim 43,wherein said material comprises a bead.
 52. The method according toclaim 51, said bead comprises a microbead having a diameter ofapproximately 30 to 300 microns.